The calcitonin receptor protects against bone loss and excessive inflammation in collagen antibody-induced arthritis

Summary Pharmacological application of teleost calcitonin (CT) has been shown to exert chondroprotective and anti-resorptive effects in patients with rheumatoid arthritis (RA). However, the role of endogenous CT that signals through the calcitonin receptor (CTR) remains elusive. Collagen II antibody-induced arthritis (CAIA) was stimulated in wild type (WT) and CTR-deficient (Calcr−/−) mice. Animals were monitored over 10 or 48 days. Joint inflammation, cartilage degradation, and bone erosions were assessed by clinical arthritis score, histology, histomorphometry, gene expression analysis, and μ-computed tomography. CAIA was accompanied by elevated systemic CT levels and CTR expression in the articular cartilage. Inflammation, cartilage degradation, and systemic bone loss were more pronounced in Calcr−/− CAIA mice. Expression of various pro-inflammatory, bone resorption, and catabolic cartilage markers were exclusively increased in Calcr−/− CAIA mice. Endogenous CT signaling through the mammalian CTR has the potential to protect against joint inflammation, cartilage degradation, and excessive bone remodeling in experimental RA.


INTRODUCTION
Rheumatoid arthritis (RA), a chronic inflammatory autoimmune disease, affects approximately 0.5% to 1% of today's population. The progressive systemic disease mainly affects symmetrical joints because of leukocyte infiltration of the synovial membrane caused by an unbalanced activation of the innate and adaptive immune system. Chondrocyte catabolism and enhanced osteoclastogenesis cause articular destruction that results in debilitating pain, joint swelling, and morning stiffness (Smolen et al., 2016).
First discovered in the 1960s, calcitonin (CT) was shown to mediate bone and cartilage turnover. Produced by parafollicular C cells of the thyroid gland, circulating CT is able to regulate osteoclast function and control the calcium and phosphate metabolism. CT binds to the calcitonin receptor (CTR), a 7-pass transmembrane protein primarily expressed in the central nervous system, the kidney, and osteoclasts. Pharmacologically employed CTR agonists, most commonly obtained from salmon or eel, exhibit a more than 50-fold higher potency than mammalian CT and have been approved by the FDA for hypercalcemic emergencies and osteoporosis treatment (Cosman et al., 2014).
Teleost CT treatment reduced systemic levels of interleukin (IL)-1 and immunoglobulins (Aida et al., 1994) and partially protected against bone erosions in patients suffering from RA (Sileghem et al., 1992). Apart from the bone sparing effects attributed to an inhibition of osteoclastogenesis and an induction of osteoblastogenesis Sondergaard et al., 2012), treatment with teleost CT achieved a reduction of pain in murine collagen-induced arthritis (Katri et al., 2019).
After initial excitement about the broad use of teleost CT for the treatment of osteoporosis in the 1980s, rather disappointing results for fracture prevention led to an almost complete withdrawal of the costly drug from the market (Chesnut et al., 2000). In respect to RA and osteoarthritis (OA), teleost CT has not advanced into clinical application up to now (Ozoran et al., 2007).
Owing to a hitherto lack of in vivo evidence for a potential physiological role of CTR signaling in RA, we compared the course of collagen II antibody-induced arthritis (CAIA) in wild type (WT) and CTR-deficient (Calcr À/À ) mice. Disease progression and resolution were assessed on functional, histological, radiological, and gene expression levels. Our results suggest a pivotal role of endogenous CT/CTR signaling with the potential to protect against excessive systemic and local inflammation during RA, in addition to preserving a physiological bone metabolism.

Arthritis increases CT serum concentrations and the CTR is expressed in the articular cartilage of knee joints
To assess the role of endogenous CT signaling in experimental RA, we first performed serum ELISA analyses, where arthritic WT CAIA animals showed significantly higher CT levels compared to healthy controls (CTRLs) on day 10 ( Figure 1A). Next, we monitored mRNA expression of Calcr in ankle joints of WT CAIA and CTRL mice, which remained comparatively constant over the course of arthritis ( Figure 1B). Immunofluorescence of knee and ankle joints of WT CAIA mice confirmed CTR expression in corresponding superficial articular tissues, where the macrophages marker CD68 was absent ( Figure 1C; Figure S1).

CTR-deficiency does not alter clinical arthritis development but is associated with excessive acute intraarticular inflammation and concomitant cartilage degradation of knee joints
Clinical signs of arthritis peaked around day 8 and resolved subsequently following the transient nature of the CAIA model ( Figure 2A). The posterior probability of a clinical difference between WT CAIA and Calcr À/À CAIA mice was 87.9% on day 8. From day 9 on, the posterior mean of WT CAIA animals dropped faster than that of Calcr À/À CAIA mice, being virtually equal on day 16 (Calcr À/À CAIA: 4.14 and WT CAIA: 4.18, with a posterior probability of WT CAIA having a larger score of 51.8%). From day 17 on, Calcr À/À CAIA animals displayed larger estimated scores, but on a generally low level. (C) Representative immunofluorescent stainings of coronal WT CAIA knee joint sections on day 10 using a CTR-specific antibody (purple) and blue nucleus stain (DAPI) (upper row), and an additional CD68-specific antibody (green) stain (lower row). Scale bars 500 mm (overview) and 50 mm (detail). Given values for serum CT are median G minimum and maximum, gene expression values are median G minimum and maximum as relative fold changes of the WT CAIA group with respect to the WT CTRL group that was set to 1. Parallel to the onset of arthritis, all animals lost body weight, before regaining weight following the clinical peak of arthritis on day 8 ( Figure 2B). Between day 4 and 35, the posterior probability for Calcr À/À mice to lose more weight than WT mice was consistently larger than 99%. On day 48, the posterior probability for a difference between groups was 80.4%.
In accordance with clinical findings, the histopathological inflammation score for knee joints was significantly increased in both WT CAIA and Calcr À/À CAIA mice compared to CTRLs on day 10. A similar tendency was observed for Calcr À/À animals on day 48 ( Figures 2C and 2D). For cartilage degradation, WT CAIA and Calcr À/À CAIA animals scored significantly higher than their CTRL groups on day 10. This was maintained in Calcr À/À CAIA animals on day 48 while only a tendency was observed in WT mice (Figures 2C and 2E). Histomorphological assessment of proximal tibiae revealed a significantly increased number of osteoclasts on day 10 for WT CAIA mice, whereas this was not the case for Calcr À/À animals ( Figures  2C and 2F).

Disrupted CTR signaling leads to pronounced inflammation and cartilage degradation in ankle joints
Histopathological analyses ( Figure 3A) of ankle joints further confirmed clinical findings ( Figure 3B), where WT CAIA and Calcr À/À CAIA mice showed significantly higher inflammation scores than their respective CTRL groups on day 10. For cartilage degradation, Calcr À/À CAIA mice scored significantly higher than their corresponding CTRL group on day 10, whereas only a tendency was observed in WT animals ( Figures  3A and 3D). The bone erosion score on day 10 was significantly higher in WT CAIA animals compared to CTRLs, whereas this observation was much less pronounced in Calcr À/À CAIA animals. As expected, no changes were observed on day 48 ( Figures 3A and 3E).

Mice lacking the CTR display impaired expression levels of cartilage and bone turnover markers
To correlate histological findings with molecular data, we performed gene expression analyses of ankle joints. WT CAIA animals showed a significantly higher expression of collagen formation markers (collagen type I alpha 1 chain (Col1a1), collagen type II alpha 1 chain (Col2a1)), both crucial for repairing damaged bone and cartilage tissue ( Figure 4A). In turn, only Calcr À/À CAIA mice showed an increased expression of proteolytic matrix metalloproteinase 13 (Mmp13) and main cartilage proteoglycan, aggrecan (Acan), on day 10. On day 48, only Col1a1 proved to be significantly elevated in Calcr À/À CAIA mice, whereas Sox9 was significantly reduced exclusively in WT CAIA animals.
Looking at bone turnover markers, we found an induced expression of bone resorption markers acid phosphatase 5 (Acp5) and cathepsin K (Ctsk) in Calcr À/À CAIA animals on day 10, followed by an increased expression of the bone formation marker Bglap (encoding osteocalcin) on day 48 ( Figure 4B). These data indicate that CTR-deficiency leads to an enhanced expression of bone resorption markers during acute arthritis, followed by an increase of bone formation markers during the resolution phase.
CTR-deficiency is associated with a severe disruption of bone integrity during acute and longterm experimental RA To further understand short-term and long-term effects of the CTR on bone remodeling during experimental RA, m-computed tomography (mCT) analyses of proximal tibiae were conducted. On day 10, bone volume, defined as the volume of mineralized bone per total volume of interest (BV/TV), bone density, and trabecular thickness (Tb.Th) were exclusively reduced in Calcr À/À CAIA mice ( Figure 5A), whereas bone surface was only reduced in WT CAIA animals. On day 48, bone volume, bone surface, and trabecular number (Tb.N) were again exclusively reduced in Calcr À/À CAIA mice, whereas trabecular separation (Tb.Sp) was coherently increased ( Figure 5A). To analyze systemic bone changes over time, we compared proximal tibiae on day 10 to those on day 48 within groups. For Calcr À/À CAIA animals, BV/TV, bone surface, and Tb.N decreased, whereas Tb.Sp increased over time, indicating a relevant loss of bone integrity over the course of 48 days ( Figures 5B-5D). Interestingly, bone density and Tb.Th increased for Calcr À/À CAIA mice over time, indicating an overall loss of bone substance, with the remaining bone being remodeled to a sclerotic phenotype, most prominent in Calcr À/À CAIA knee joint samples on day 48 ( Figure 5D). Lastly, increased bone density was also    iScience Article observed for WT CAIA and WT CTRL animals, mimicking some degree of bone deposition and remodeling over time (Figures 5B and 5C).
Inflammation and immunomodulatory costimulatory molecule expressions are predominantly increased in arthritic CTR-deficient mice Finally, we conducted gene expression analyses of inflammatory markers in ankle joints. Although the expression of Tnfa increased in WT CAIA and Calcr À/À CAIA mice, sphingosine kinase-1 (Sphk1), catalyzing the conversion of sphingosine to sphingosine-1-phosphate (S1P) and thus enhancing TNF-a signaling, was only overexpressed in Calcr À/À CAIA mice on day 10 and day 48 ( Figure 6A). Expressions of Tgfb, Il1b, and Ccl2 were also exclusively increased in Calcr À/À CAIA animals on day 10. Corresponding to clinical results, no differences in the expression of respective genes were observed on day 48, except for Sphk1. Lastly, expressions of Cd14 and Cd68, both macrophage surface markers, were only elevated in Calcr À/À CAIA mice ( Figure 6).

DISCUSSION
Pharmacological applications of teleost CT were previously proposed to prevent bone defects in RA (Aida et al., 1994;Ozoran et al., 2007), osteoporosis (Kaskani et al., 2005) and OA (Karsdal et al., 2015). With our current study, we unveil an independent bone and cartilage sparing, anti-inflammatory and immunosuppressive role of the mammalian CTR in antibody-mediated arthritis.
Employing the CAIA model, we were previously able to demonstrate a dual pro-inflammatory and bone protective role of the neuropeptide calcitonin gene-related peptide alpha (aCGRP) (Maleitzke et al., 2020). Although different in structure, both CT and aCGRP are encoded by the common gene Calca and synthesized through alternative splicing. Similar to aCGRP, the data presented herein suggest an indispensable role of the CTR in the preservation of bone integrity during inflammatory arthritis. Although aCGRP however acts as a pro-inflammatory peptide during acute experimental arthritis, we show that the CTR protects articular structures from excessive inflammation. Teleost CT was previously shown to have a high in vivo potency (Gydesen et al., 2016) as well as antiresorptive and chondroprotective properties in the treatment of experimental RA and OA (Karsdal et al., 2008;Sondergaard et al., 2012;Wen et al., 2016), yet little is known about the role of endogenous CT/CTR signaling in inflammatory joint diseases.
Contrary to the reported osteoprotective effects of teleost CT through an inhibition of osteoclast activity (Karsdal et al., 2008), our group could previously show that endogenous CT suppresses bone formation through the inhibition of S1P secretion from osteoclasts (Keller et al., 2014). Accordingly, our current findings indicate that CTR signaling is indispensable to control and suppress the expression of Sphk1 during acute and chronic arthritis. Sphk1 enzymatically generates S1P, a molecule coupling bone formation to resorption (Martin and Sims, 2015), while maintaining an inflammatory response in RA, where it is found elevated in the synovial fluid of affected patients (Hu et al., 2011;Lai et al., 2008). Inhibition of S1P led to decreased serum levels of pro-inflammatory TNF-a, IL-1b, and IL-6 and reduced arthritis activity in previous in vivo experiments (Lai et al., 2008;Alvarez et al., 2010), allowing the assumption that S1P is a key regulator of TNF-a stimulated IL-1b and IL-6 release in RA. Congruently, we found CTR deficient mice to display significantly elevated expression levels of Sphk1, Tnfa, and Il1b during acute and chronic arthritis.
Elevated TGF-b1 expression levels are found in synovial fibroblasts of RA patients and a blockade of the multifunctional cytokine has been proposed as an experimental RA treatment approach (Pohlers et al., 2007;Sakuma et al., 2007). In this study, inactivated CTR signaling led to an increased Tgfb1 expression, underlining the anti-inflammatory effect of the CTR in antibody-mediated arthritis.
In joints, collagen synthesis is primarily dependent on Col1a1 and Col1a2 expression, both essential for the formation of bone and cartilage, respectively. Although WT CAIA animals showed strongly enhanced Col1a1 and Col1a2 expression levels during acute arthritis, this repair mechanism was not observed in Calcr À/À CAIA mice. Only as arthritis subsided, Col1a1 was increased in Calcr À/À CAIA mice. Accordingly, iScience Article CT treatment was previously shown to increase proteoglycan and collagen type II synthesis in human OA cartilage (Sondergaard et al., 2006(Sondergaard et al., , 2010. Alongside collagen synthesis marker suppression, the expression of Mmp13 was congruently elevated in mutant animals during acute arthritis. Previous data showed intraarticular salmon CT to reduce arthritis in vivo and suppress the expression of MMP1, 3 and 13, all enzymes enhancing collagen fiber degradation (Ryan et al., 2013). Contradictory to our findings, CT was Sphk1 Figure 6. The CTR is essential to contain excessive expression of inflammation markers qRT-PCR expression analyses of inflammation and immunomodulation genes of ankle joints on day 10 and 48. Given values are median G minimum and maximum as relative fold changes of WT CAIA and Calcr À/À CAIA groups with respect to CTRL groups that were set to 1.

OPEN ACCESS
iScience 25, 103689, January 21, 2022 9 iScience Article previously reported to enhance the expression of aggrecan (El Hajjaji et al., 2004), which can however also occur as a response to arthritic cartilage erosion and subsequent aggrecan degradation (Roughley and Mort, 2014).
In line with our previous findings regarding the function of mammalian CT as a physiological inhibitor of bone formation (Keller et al., 2014), we found the expression of the key osteoblast transcription factor Runx2 to be significantly enhanced in Calcr À/À CAIA mice, accompanied by increased expression levels of osteoclast markers Acp5 and Ctsk. This indicates that during RA, the CTR is required to limit excessive bone turnover as a whole, rather than bone formation or resorption singularly. As a result, systemic bone integrity decreased markedly in Calcr À/À CAIA mice over time.
Although aCGRP was previously shown to induce the production of CCL2 (Malon et al., 2011), a pro-inflammatory chemokine regulating monocyte migration in RA and OA (Raghu et al., 2017), no such data exist on CT or CTR up to now. In our study, we found that disrupted CTR signaling led to an increased expression of Ccl2, underlining the anti-inflammatory potential of the CTR in experimental RA. Although a first randomized controlled trial from 2006 could not show a detectable clinical benefit for CCL2 antibody therapy in RA (Haringman et al., 2006), targeting CCL2 is still being extensively discussed as a novel RA treatment approach (Moadab et al., 2021;Zhang et al., 2015;Chen et al., 2017). As CD14 (Park et al., 2016) and CD68 (Bresnihan et al., 2009) have both been proposed as biomarkers for the therapeutic response in patients with RA, our findings of increased Cd14 and Cd68 expression levels in Calcr À/À CAIA mice, suggest a mechanistic involvement of the CTR in controlling the overexpression of these monocyte/macrophage surface markers.
The deduced functions of the CTR not only include a potent anti-inflammatory effect in affected joints, but also a protective role regarding cartilage and bone degradation during RA. Together, these results encourage further exploration of CTR signaling in RA patients, as the CTR represents a G-protein-coupled cell surface receptor known as an excellent drug target.

Limitations of the study
There are certain limitations to this study. First, although the CAIA model exhibits several similarities to human RA, its initiation works through preformed autoantibodies mostly independent of T-cells and B-cells, which are highly relevant in human RA (Nandakumar et al., 2004). Moreover, although the murine CAIA model affects joints symmetrically similar to human RA, its transient nature is different from the chronic disease progression in affected patients. Despite these limitations, we believe that our findings are especially important from a clinical point of view, as novel water soluble CT prodrugs have recently been introduced for the treatment of osteoporosis and related musculoskeletal disorders like RA (Renawala et al., 2021).

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following: iScience Article facilitate CAIA development in 15 Calcr À/À CAIA and 13 WT CAIA mice. WT CTRL (n = 8) and Calcr À/À CTRL mice (n = 8) received respective i.p. injections of sterile phosphate buffered saline (PBS). The ArthritoMab antibody cocktail is based on monoclonal antibodies to collagen type II, causing a complement-dependent inflammatory joint reaction (Nandakumar et al., 2003). The CAIA model is sex-and age-dependent, with male and aged animals being more susceptible to CAIA. A protective role of estradiol against CAIA is being discussed (Nandakumar et al., 2003). To ensure arthritis onset and progression, male mice aged 10-12 weeks were chosen for all experiments.

Study design
All animals were monitored daily over the course of 10 or 48 days. To assess the acute inflammatory phase of arthritis, 8 Calcr À/À CAIA and 8 WT CAIA animals in addition to 4 Calcr À/À CTRL and 4 WT CTRL animals were euthanized on day 10. To investigate the chronic or resolution phase of arthritis, 7 Calcr À/À CAIA and 5 WT CAIA, 4 Calcr À/À CTRL and 4 WT CTRL mice were euthanized on day 48.

Arthritis score, weight, humane endpoints
Arthritis was assessed daily, following weighing of animals, employing a semiquantitative clinical arthritis score (Lee et al., 2006) by two blinded observers (T.M. and A.H.). A score between 0 and 3 was given based on redness, swelling and number of affected digits for each paw and scores were added up (maximum score of 12). 0: No swelling; 1: Mild to moderate swelling and erythematic ankle and/or 1 swollen digit; 2: Moderate swelling and erythematic ankle or swelling in 2 or more digits; 3: Marked swelling along all aspects of the paw or all 5 digits swollen.
Weight loss of >30% compared to baseline without recovery within 24h, limping and avoidance of movement and grooming were defined as humane endpoints. The clinical arthritis score served as the primary experimental outcome. All other parameters served as secondary outcomes.

Sample preparation
Left ankle joints were carefully dissected, stripped of all muscle and soft tissue and snap frozen in liquid nitrogen for RNA isolation and gene expression analysis. Right ankle and knee joints were fixed in paraformaldehyde (PFA) 4% before mCT analysis. After radiologic analysis, samples were embedded in SCEM medium as previously described (Kawamoto and Shimizu, 2000).

Histology
Embedded samples were stored at À80 C and serial sections of 7 mm were cut using a CM3050 S Kryostat cryo microtome (Leica Biosystems, Wetzlar, Germany). Central coronal views of knee joints and central lateral views of ankle joints were obtained and sections were stained with 1) hematoxylin and eosin (H&E), 2) toluidine-blue and 3) tartrate-resistant acid phosphatase (TRAP). A previously established scoring system from 0 to 3 was applied by two blinded investigators (T.M. and A.H.) (Tu et al., 2016;Bendele et al., 1999).
H&E: Inflammation score: 0: normal; 1: mild inflammatory infiltration with no soft tissue edema or synovial lining cell hyperplasia; 2: moderate infiltration with surrounding soft tissue edema and some synovial lining cell hyperplasia; 3: severe infiltration with marked soft tissue edema and synovial lining cell hyperplasia.
TRAP: Bone erosion score: 0: normal; 1: mild (some areas of resorption not readily apparent on low magnification with visible osteoclasts); 2: moderate (obvious bone resorption with a few osteoclasts visible); 3: marked (large erosion areas extending into the bone cortex with numerous osteoclasts visible in all areas).

Quantitative real time polymerase chain reaction (qRT-PCR)
Snap-frozen ankle joint samples were trimmed to their corresponding tibiotalar articular surfaces. This resulted in samples including cartilage, small amounts of synovium, periarticular tissue, and minimal bone tissue. Samples were homogenized using an Ultra Turrax disperser (IKA-Werke, Staufen, Germany) with Trizol (Thermo Fisher Scientific, Waltham, MA, USA). RNA was isolated with the RNeasy mini Kit (Qiagen, Hilden, Germany), including DNase I treatment. Concentrations were determined and purity monitored (A260/A280 ratio of 1.9-2.1) using a NanoPhotometer P360 (Implen GmbH, Munich, Germany). 0.25-1 mg of RNA was reverse transcribed to complementary DNA (cDNA) using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). qRT-PCR was performed on 384 well-plates in a 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific), employing Sequence Detection System (SDS) software (Thermo Fisher Scientific). The Power SYBR Green PCR master mix (Thermo Fisher Scientific) was run at an annealing temperature of 60 C with 2 ng of cDNA per well. For each run threshold cycle (Ct), and melting curve were calculated, confirming PCR product specificity.
Primers were individually designed using the GRCm38.p6 C57BL/6J reference genome and employing Primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/), spanning at least two exons with a large intron in between to avoid genomic DNA amplifications. Primers were obtained from Eurofins Genomics GmbH (Luxemburg) and used at a final concentration of 0.2 mM. Gene expressions were normalized per housekeeping gene, glycerinaldehyde-3-phosphate-dehydrogenase (Gapdh), and values for WT CAIA and Calcr À/À CAIA groups are displayed as fold changes relative to respective CTRL samples.